The present invention relates to a method for preparing decorated macromolecular scaffolds. More particularly, the invention relates to a method for preparing molecular scaffolds decorated with ligands and with a high degree of decoration. The method of the invention is useful for the generation of bioactive nanoparticles for use in clinical applications. Such applications include drug and gene delivery, tumour targeting, bioimaging, tissue remodelling, generation of antiviral products and vaccines delivery.
Bioactive nanoparticles are nanoparticles carrying biologically active ligands (for example drugs, peptides, and vaccines). Bioactive nanoparticles are much smaller than human cells but are similar in size to large biomolecules such as enzymes and receptors and can therefore easily enter most cells, and move in and out of blood vessels enabling them to circulate through the body. Bioactive nanoparticles are attractive candidates for use as targeted drug delivery vehicles. Bioactive nanoparticles are based on nanoparticles such as liposomes, micelles, macromolecular scaffolds such as dendrimers, nanolipospheres, silica-coated micelles and ceramic nanoparticles.
Bioactive nanoparticles comprised of random polymers having ligands conjugated onto the random polymer can be readily produced. However the conjugation of ligands onto macromolecular scaffolds, for example dendrimers, has proved to be more difficult.
Dendrimers are macromolecular scaffolds with a well-defined highly branched structure carrying a number of reactive surface groups. The number of surface groups on a macromolecular scaffold is referred to as the valency of the macromolecular scaffold. The highly branched structure is the result of an iterative synthetic process starting from a central core which is extended outwards by a series of reactions which result in the branching structure. The scheme below represents a central core with a valency of four. Each iterative synthetic step doubles the number of surface groups.

The number of branch points encountered upon moving outwards from the core to the surface groups of the dendrimer define the generation of the dendrimer. G-0 refers to a generation zero dendrimer. Thus, in scheme 1, the G-0 dendrimer has four surface groups (i.e. a tetramer), the G-1 surface group has eight surface groups (i.e. an octamer) and the G-2 dendrimer has 16 surface groups. In this example, the number of surface groups doubles with each branching step, but introduction of a trivalent branching molecule would treble the number of surface groups, etc. As the molecular weight and generation increases, the dendrimer tends to become more globular or spherical and the surface groups tend to become more closely packed. For a review of dendrimers their properties and applications see Klajnert et al. 2001, Vol. 48, 199-208.
Poly(amidoamine) (or PAMAM) dendrimers are one of the most well known types of dendrimers and are commercially available. The PAMAM dendrimers have branches comprised of branching amidoamine units (—CH2CH2C(O)NHCH2CH2NH2) and are available with a variety of different cores and surface groups. The surface groups are the functional groups at the end of each branch of the dendrimer. In the case of PAMAM the surface group is usually an amino group. However, PAMAM dendrimers are available with alternative surface groups for example alcohol groups (commercially available and often referred to either PAMAM-OH or PAMAM-amidoethanol).

Other types of dendrimer include polyamine dendrimers (branches comprised of repeating amine units), polyimide dendrimers (branches comprised of repeating amide units), polypropyleneimine (PPI) dendrimers also known as polypropylamine (POPAM) dendrimers (branches comprised of repeating propylamine units), dendrimers based on poly(arylether) units and multiple antigen peptide (MAP) dendrimers which are based on repeating polylysine units.
A macromolecular scaffold may be decorated by conjugating ligands via the surface groups of the macromolecular scaffold. The number of ligands that can be conjugated onto a random polymer is relatively low compared to number of ligands which can potentially be conjugated to a well-defined highly branched macromolecular scaffold such as a dendrimer. Bioactive nanoparticles based on dendrimers therefore provide a means to achieve concentrated payloads of the active ligand.
In summary, dendrimers are particularly attractive scaffolds for the generation of bioactive nanoparticles for clinical applications because their size, structure and properties can be manipulated to suit its application and in particular because dendrimers can carry multiple ligands per molecule.
Unfortunately, the current approaches used to conjugate ligands onto random polymers are not powerful enough to enable more than partial ligand decoration of macromolecular scaffolds such as dendrimers. Problems occur from side reactions and incomplete reactions of the surface groups of the macromolecular scaffold which lead to structural defects. For clinical applications it is highly desirable to obtain a homogeneous and high degree of decoration, preferably fully decorated scaffolds, thereby maximizing the possible benefits to biological activity of multivalent display as well as ensuring homogeneity and reproducibility from batch to batch and within each batch.
Chemoselective ligations and bioconjugations are used to link complex or precious molecules and there are many different ligation techniques, including cycloadditions, the Staudinger ligation, olefin cross metathesis, native chemical ligation and hydrazone and oxime ligations. Attempts to apply some of these ligation techniques to dendrimers have been made. However, these techniques are generally not powerful enough to fully decorate dendrimers more complex than tetramers (i.e. a dendrimer with four surface groups) and octamers (i.e. a dendrimer with eight surface groups).
Oxime ligations (i.e. conjugation of two moieties via an oxime bond) have been used to conjugate biological molecules because the oxime bond is stable under physiological conditions. In particular, oxime bond forming condensation reactions between compounds bearing carbonyl groups and hydroxylamino nucleophiles have proven useful for the formation of a number of bioconjugates. However, the utility of these reactions in the formation of bioconjugates, and particularly in the formation of bioconjugates having multiple oxime bonds (i.e. polyoximes), has been hindered by the slow reaction rate at neutral pH.

Rose et al. (Bioconj. Chem., 1996, 7, 552-6) investigated conditions for the formation of oxime bonds between peptides and the stability of these bonds at various pH. In particular, Rose et al. studied oxime bond formation as a function of pH. In the case of single oxime bond formation, the condensation reaction was found to go essentially to completion at pH 3.0 and 4.6 but was markedly faster at lower pH. The reaction at pH 5.3 did not reach completion after 24 hours. In the case of polyoxime bond formation (i.e. formation of multiple oxime bonds on a single species), formation of the polyoxime product (a hexaoxime product in the investigation carried out by Rose et al.) was fastest at pH 2.1 and somewhat slower at pH 4.6. The oxime bond forming reaction did not take place at all at pH 7.0. Even at optimally low pH (i.e. 2.1), the major product was the penta- and not the hexaoxime, thus reflecting the limitations of the oxime bond forming reaction when applied to situations where multiple oxime bonds are being formed.
Dirksen and co-workers (see Dirksen et al. Angewandte Chemie, 2006, 45, 7581-7584) have investigated the oxime ligation between a glyoxylyl-functionalised peptide and an aminooxyacetyl-functionalised peptide and shown that the single oxime bond forming reaction can be catalysed using aniline at pH 4.5. The aniline catalyst was found to speed up the conjugation reaction rate but did not change the equilibrium position for the oxime ligation. Thus, the reactions investigated by Dirksen et al. eventually reached 99% conversion regardless of whether an aniline catalyst was employed or not.
Other efforts to improve the oxime ligation between functionalised peptides include utilising peptides functionalised with pyruvic acid in place of peptides functionalised with levulinic acid (see Kochendoerfer et al. Bioconjugate Chem., 2002, Vol 13, 474-480). The pyruvic acid ketoxime bond is believed to be resonance-stabilized by conjugation with its carbonyl group and therefore it is thought that the oxime bonds formed in these ligations would be more stable than the related levulinic acid oxime.

Oxime ligations have also been applied in the formation of peptide and carbohydrate dendrimers (see Mitchell et al. Bioorg Med Chem Lett., 1999, 2785-2788). However, as noted above while oxime ligation has proven to be useful in the formation of bioconjugates having a single oxime bond (for example in the formation of an oxime bond between two peptides), use of oxime ligation to decorate dendrimers (requiring the formation of multiple oxime bonds) with ligands has proved to be more challenging. Mitchell et al. found that even at G-1 PAMAM (a commercially available polyamidoamine dendrimer with eight amino surface groups) only partial decoration could be achieved. Various different reaction conditions were investigated by Mitchell et al. yet all attempts to fully decorate the dendrimer failed.
To date, one of the most successful methodologies for decorating dendrimers involves a native chemical ligation between a cysteine residue attached to the surface groups of a dendrimer and a peptide ligand prepared with a C-terminal thioester. Baal and co-workers (Angew, Chem. Int. Ed, 2005, 44, 5052-5057) reported successful decoration of G-2 PAMAM (i.e. a dendrimer with a valency of 16) using this method, although the batch of decorated dendrimer appeared to contain partially decorated products.

Another promising strategy which has been used for the preparation of decorated dendrimers employs “click chemistry”. Specifically, reactions such as the azide-alkyne cycloadditions, have been investigated. Chun and co-workers utilised a copper-catalyzed cycloaddition reaction to conjugate peptides functionalised with azide groups to G-0 PAMAM dendrimers (i.e. a tetramer), modified with terminal alkyne groups (Australian Journal of Chemistry, 62, 1339-1342). A further example utilising “click chemistry” has been applied to a non-symmetrical dendrimer by Wu and co-workers (Chem Commun., 2005, 5775-5777). Wu and co-workers achieved decoration of a scaffold with eight surface groups.
The decoration of dendrimer with more than eight surface groups still remains a challenge. Higher generation dendrimers are thought to adopt a conformation wherein the surface groups are shielded. For example, when there is a lack of binding interactions between both the dendrimer branches and the surface groups on dendrimers the dendrimer branches have high mobility and can fold inwards (see Boas et al. Dendrimers in medicine, 2006, 1-27). This back-folded conformation is believed to be more prevalent in higher generation dendrimers. Also, dendrimers (such as PAMAM and PPI dendrimers) which have basic surface groups as well as a basic interior are thought to be sensitive to pH. For example at low pH electrostatic repulsion between positively charged ammonium groups within the interior of dendrimers with amine containing branches are thought to result in an extended conformation (see Boas et al. Dendrimers in medicine, 2006, 1-27).

In summary, various ligation reactions have been employed in the conjugation of ligands to macromolecular scaffolds. Complete decoration of G-2 PAMAM has been reported using native ligation. However, the batch was not homogeneous. Only partial decoration of G-1 PAMAM has been achieved using oxime ligations and there remains a need to provide a method for generating highly decorated dendrimers, particularly those of higher generations. It is also desirable to provide a method which gives highly decorated dendrimers as a homogenous batch.